Salmonella typhimurium is a common cause of foodborne enterocolitis in the U.S. and developing countries. The clinical manifestations of S. typhimurium infection result from the presence of polymorphonuclear leukocytes (PMN) in the intestinal mucosa and lumen that are recruited along a gradient of chemotactic chemokines such as interleukin-8. These inflammatory mediators are activated at the transcriptional level by the action of DNA-binding transcription factors, of which NF-kappaB is the key member. We have described strains of human non-pathogenic Salmonellae that inhibit activation of inflammatory processes in vitro. Infected or colonized epithelial cells become refractory to inflammatory responses elicited by pathogens and cytokines because of inhibitory effects on the NF-kappaB signal transduction pathway. This inhibition appears to result from a block of IkappaBeta-a ubiquitination but not phosphorylation. We have identified a candidate bacterial protein, AvrA, which we hypothesize mediates this phenomenon. The goal of this proposal is to identify mechanisms by which bacteria inhibit ubiquitination to block NF-kappaB activation. The specific aims in this proposal are 1) to determine the mechanism of AvrA inhibition of NF-kappaB activation; 2) to define the biological role of AvrA in epithelia; and 3) to characterize other mechanisms by which Salmonella alter ubiquitination in epithelial signaling pathways. In these studies, we will examine 2 mechanisms that Salmonella or its effector protein AvrA may use to inhibit NF-kB activation: deubiquitination of IkappaBeta and interference of IkappaBeta ubiquitin ligase. We will use transfection and infection methods to introduce AvrA into epithelial cells to study the biological effects of AvrA on proinflammatory and apoptotic pathways. We will use novel biochemical reagents to determine if AvrA targets ubiquitin or ubiquitin-like modifications. Such studies will provide insights into the molecular basis of bacterial inhibition of ubiquitination in epithelial cell signaling pathways which may lead to therapeutic inventions in the treatment of acute inflammatory diseases of mucosal tissues.